Insect ecdysis sequences represent a simple, robust, and tractable model for studying the neuromodulatory mechanisms that govern behavior. Because initiation of an ecdysis sequence involves a profound shift in behavioral priorities, study of these sequences offers the opportunity to understand the neuromodulatory mechanisms that govern changes in behavioral state. In addition, because ecdysis behaviors are inherently sequential, they permit the systematic investigation of how motor programs are assembled and serially executed by the nervous system. Finally, the study of ecdysis sequences promises insight into how conserved circuits can be variably configured to generate immensely different behaviors. In Drosophila, for example, the motor sequences performed at pupal and adult ecdysisbefore and after metamorphosis, respectivelyare scarcely similar though they are governed by a common set of neuromodulatory/hormonal inputs. By analogy to computing, these inputs can be regarded as instructions written in a higher programming language that are then compiled into different outputs. Exposing the mechanisms of neural compilation in ecdysis is likely to deeply inform our understanding of how neuromodulators contribute to neurocomputation by reconfiguring the activity of neural networks. To investigate these questions, my laboratory seeks to elucidate the circuitry that governs both the pupal and adult ecdysis sequences in Drosophila. Our efforts over the last year have been more or less evenly divided between study of these two circuits. In addition, we have devoted considerable effort to developing a novel methodology for genetically targeting small subsets of neurons in the fly brain for manipulation. Such methods are required to facilitate the fine-mapping of neuronal circuits by our lab and others. Each of the three projects pursued in 2018 had a significant time horizon and each is only now being written up for publication. With regard to pupal ecdysis, the work conducted over the past year departs from our typical focus on neural circuitry and involves more carefully describing the behavior being generated by the brain. This shift in focus stemmed from the realization that if we are to understand in detail how the fly brain generates a pupal ecdysis sequence, we must first understand in detail what constitutes that behavior. The appropriate level of description for this purpose is that of the individual muscle contractions that produce the movements executed by the animal during performance of the pupal ecdysis sequence. Accordingly, we have developed tools to comprehensively monitor the activity of the entire pupal musculature during ecdysis. We use genetically-encoded Ca++ indicators to generate time-series data of muscle activity. These data define the ecdysis sequence in terms of all the individual muscle contractions. Achieving this objective has required mapping the entire pupal musculature and its pattern of innervation. In addition, to confirm that muscle contractions are neurally driven, we have developed methods for simultaneously imaging the activity of both muscles and their synaptic inputs. To determine which inputs are subject to modulation by Crustacean Cardioactive Peptide (CCAP), as described in our 2017 eLIFE paper (Diao et al., doi: 10.7554/eLife.29797), we have identified the complement of motor neurons that express the CCAP receptor. This work is currently being prepared for publication. Our study of the adult ecdysis circuit has focused on the neural mechanisms that mediate the environmentally-sensitive decision to delay wing expansion under adverse conditions. Our previous work had shown that this decision was likely mediated by two neurons (i.e. the BSEG) that secrete the hormone Bursicon (Luan et al., 2012, J Neurosci. 32: 880889). Using the Trojan exon technology that we developed in 2015 (Diao et al., 2015, Cell Rep. 10:1410-21. doi: 10.1016/j.celrep.2015.01.059), we have characterized downstream targets of the BSEG and found, surprisingly, that they include a set of cholinergic neurons that signal back to the BSEG via a positive feedback loop. This feedback loop is critical for initiating and maintaining wing expansion and thus represents a key component of the decision-making circuitry. This work is likewise being prepared for publication. The novel technology that we have developed during the past year involves the use of split inteins, which are recently discovered proteins that can join two protein fragments fused to them. Split inteins lend themselves naturally to applications in which a protein molecule is divided into inert fragments that must be joined together to reconstitute activity. Our method divides the Cre recombinase into three fragments fused to two distinct pairs of split inteins. The three components can thus be independently targeted to different cell groups and only those cells that express all three Cre components will generate active Cre. Cre itself is used to activate a transcription factor, Gal4, thus allowing expression of other transgenes in the targeted cells so that they can be manipulated to determine their function. A manuscript describing this methodology is currently being prepared for submission, but we have already shared our unpublished reagents with numerous colleagues to facilitate their circuit-mapping efforts. In summary, we have made good progress during the last year in advancing research on the principal questions of interest to the laboratory. At the same time, we have continued to contribute tools that support not only our own circuit mapping efforts, but also those of other members of the Drosophila research community. As we use these tools to extend and refine our analysis of the circuitry underlying ecdysis sequences, our work should provide insight into the principles that govern the development and function of behavioral circuits in all organisms, including humans.